Classical and molecular cytogenetics in oncology Kyra Michalova and Zuzana Zemanova Center of Oncocytogenetics Institute of Clinical Biochemistry and Laboratory Diagnostics General University Hospital and First Faculty of medecine, medecine, Charles University Prague

Cytogenetic examination Clinical cytogenetics → determination of karyotype of patients with inborn deffects Prenatal diagnostics → examination of chromosomal complement of embryos embryos,, in vitro fertilization Oncocytogenetics → specification of diagnosis and prognosis of malignant diseases Services of hygiene laboratories - testing of mutagenicity of chemicals on human organism on chromosomal level, level, radiation cytogenetics etc. etc.

Chromosomal aberrations Changes of number or structure of chromosomes I. Inborn (constitutional constitutional)) aberrations are fundamental for origin of syndromes due to anomalies of chromosomes (for example Down Down´´s sy sy)); changes of chromosomes are present in all cells of the body. body. II. II. Acquired changes of chromosomes in malignant cells;; they have clonal character (only certain cellular cells clones are involved involved)).

Cancer cells high

genome

instability

-

one

of

the

most

important events in the malignant process gene

mutations

and

numerical

and/or

structural

chromosomal aberrations

Nonrandom chromosomal aberrations are associated with specific disease subtypes and have a clear prognostic implications. implications

Methods

Conventional cytogenetic analysis

mFISH

I-FISH

mBAND

WCPWCP -FISH

CGH

Array CGH

Conventional cytogenetic analysis

Karyotype analysis Detection of chromosomal aberrations (numerical x structural structural))

krev, kostní dřeň

medium

kolchicin

hypotonie

fixace

preparace

inkubace při 37°C

barvení

Postup při kultivaci a přípravě preparátů k cytogenetickému vyšetření

Chromosomal banding: banding:

Limitations of classical cytogenetics sensitivity of chromosomal banding techniques is limited these techniques require a high rate of dividing cells with good chromosomal morphology (resolution limit of 10 Mb) Mb) in some leukemias malignant cells are not proliferating in the cell culture (only the normal cells are dividing) → the results of cytogenetic examination is not representative for malignant process chromosomal changes in leukemic cells are very often complex or could be cryptic under the limit of light microscopy

Fluorescence in situ hybridization (FISH) permits detection of selected acquired genetic changes in dividing (metaphase metaphase)) and nondividing (interphase nuclei nuclei)) cells is useful in establishing the percentage of neoplastic cells at the time of diagnosis and after therapy FISH studies are used to investigate the origin and progression of hematologic malignancies and to establish which

hematopoietic

neoplastic processes

compartments

are

involved

in

Fluorescence in situ hybridization (FISH)

Cytogenetický preparát

Dvouřetězcová značená DNA

Denaturace buněčné DNA fixované na cytogenetickém preparátu

Denaturace DNA sondy

Hybridizace DNA sondy ke komplementárním úsekům cílové DNA fixované na cytogenetickém preparátu

Analýza fluorescenčních signálů ve fluorescenčním mikroskopu

Probes for specific chromosomal structures:: structures α-satellite DNA - centromeres Determination of numerical aberrations aberrations,, identification of the origin of cenromeres in marker marker--chromosomes, chromosomes, specification of cells after bone marrow transplantation (opposite sex of donor and recipient) metaphase x interphase

Locus specific DNA probes: probes: Mapping of genes on chromosomes chromosomes,, detection of structural rearrangements (translocations translocations,, deletions) deletions) metaphase x interphase

Chromosome painting probes probes:: They contain sequences from whole chromosomes or chromosomal parts (partial probes) probes) Determination of structural rearrangements (translocations and deletions of large extent), extent), identification of origin of marker marker-chromosomes Metaphase only

Multicolor FISH - mFISH allows in one hybridization experiment distinguish according to different color every pair of autosomes and sex chromosomes and then it is possible to make analyses of the whole genome and every structural and numerical rearrangement

Multicolor FISH - mFISH analyses of complex chromosomal rearrangements in bone marrow cells of patients with hematological malignancies will bring us detailed informations about involvement of specific chromosomes or their regions into rearrangements

Multicolor banding with high resolution - mBAND enables determination of exact breakpoints of chromosomal aberrations with much higher resolution than classical banding

mBAND 1

mBAND 11

ArrayArray -based comparative genomic hybridization (aCGH aCGH)) new tool to search for recurrent gains or loss of chromosomal regions throughout the genome according to detection with very high resolution of copy number changes at DNA level only recently is aCGH successfully utilised in diagnostics of leukemias and the results revealed a large spectrum of genomic imbalancies, imbalancies, including novel recurrent deletions and amplifications

BAC arrays ∼1MB Oligo arrays ∼100 kb (maximal resolution ∼ 35 kb) kb)

The impact of conventional and molecular cytogenetic analysis in oncohematology Is part of the work up at diagnosis Provides comprehensive information on the karyotype




help to specify diagnosis




help to determine the prognosis




help monitor effectiveness of treatment

Chronic Myeloid Leukemia (CML CML)) Acute Myeloid Leukemia (AML (AML) Acute Lymphoblastic Leukemia (ALL)

Hematological malignancies

Myelodysplastic Syndromes (MDS (MDS)) Chronic Lymphocytic Leukemia (C (CLL LL)) Multiple Myeloma (MM) Non Hodkin Lymphoma (NHL)

Recommended examination procedures - depending on the type of disease and treatment protocol

CML

Chronic Myeloid Leukemia (CML)




1515-20% 20% of all leukemia cases Mainly in adults (median age 65 years years)) TriTri -phasic disease: disease: CP - chronic phase (relatively benign benign,, 3-9 years) AP - More malignant accelerated phase BC - Terminal blast crisis





Blasts increase rapidly (crowd out healthy cells cells))




One of the first malignancies in which a therapy targeting the underlying molecular defect has improved the clinical outcome of patients:: Gleevec™ patients leevec™ (Novartis) used for therapy




t(9 t(9;22)(q 22)(q34 34;;q11) 11) in 9090-95% 95% of patients (detectable by CC CC)) BCR/ABL fusion gene

One of the bestbest-studied malignancies - has served as a paradigm for elucidation how genetic changes cause cells to become malignant: malignant: Ph chromosome t(9 t(9;22)(q 22)(q34 34;;q11) 11) - BCR/ABL fusion

-

Conventional Cytogenetics: BM 24h/48h cultivation (adults) at least 20 metaphases

normal karyotype or insufficient metaphases or normal karyotype without t(9;22)

FISH: BCR/ABL1

aberrant karyotype

targeted FISH or other molecular cytogenetic techniques (mFISH/mBAND, array CGH)

Report according to ISCN

CML

Chronic Myeloid Leukemia (CML)


Heterogenous group

of malignant diseases of haemopoiesis


Accumulation

of immature myeloid cells (myeloblasts) in bone


Diagnosed in

all age groups

marrow


Most

commonly affects people older than 60 years (median age 64-68 years)


Secondary AML,

therapy-related AML


Aggressive disease - median OS 2-3
Specific chromosomal aberrations significance → stratification cytogenetic findings

of

monts with clear prognostic therapy according to

AML

Acute Myeloid Leukemia (AML)

Chromosomal aberration

Genes

Prognosis

t(8;21)(q22;q22)

RUNX1-RUNX1T1

good

inv(16)(p13.1q22) or t(16;16)(p13.1;q22)

CBFB-MYH11

good

t(15;17)(q22;q12)

PML-RARA

good

t(9;11)(p22;q23)

MLLT3-MLL

intermedial

t(6;9)(p23;q34)

DEK-NUP214

poor

inv(3)(q21q26.2) or t(3;3)(q21;q26.2)

RPN1-EVI1

worst

t(1;22)(p13;q13)

RBM15-MKL1

good

Rearrangements of MLL gene

MLL

poor

monosomy 7 or deletion of 7q31

poor

deletion of 5q31

?

poor

complex chromosomal aberrations

?

worst

AML

Cytogenetic findings in AML

AML

Prognostic impact of specific chromosomal aberrations in acute myeloid leukemia (AML) 1.0

Kumulative surviving

0.9 0.8

normal karyotype

0.7

complex karyotype

0.6

t(8;21)(q22;q22) t(15;17)(q22;q11)

0.5

inv(16)/t(16;16)

0.4

5q-

0.3

-7/7q-

0.2

+8

0.1

> 50 chromosomes

0.0 0

50 100 150 200 250 300 350 400 450 500 Time (weeks)

Alert 2006

Conventional Cytogenetics: BM 24h/48h cultivation (adults) at least 20 metaphases

aberrant karyotype

normal karyotype or insufficient metaphases

Subtype specific FISH: MLL, 5q31/5p15, 7q31/7, 8/9 PML/RARA, AML1/ETO, CBFB

targeted FISH or other molecular cytogenetic techniques (mFISH/mBAND, array CGH)

Report according to ISCN

AML

Acute Myeloid Leukemia (AML)




Accumulation of malignant, immature lymphoid cells in the bone marrow and mostly also in peripheral blood




Most common malignancy in children – representing 25% of pediatric cancer





80% of pediatric leukemias




Chromosomal aberrations: found up to 90% of cases one of the most important prognostic factors!!




Conventional chromosomal analysis are limited by the low mitotic activity of malignant B-lymphocytes → I-FISH for the most common recurrent chromosomal aberrations

Heterogenous implications

disease

-

distinct

therapeutic

and

prognostic

ALL

Acute Lymphoblastic Leukemia (ALL) Overview

Cytogenetic findings in ALL Risk Group Low risk

Cytogenetic finding High hyperdiploidy (51-65 chromosomes)

ETV6-RUNX1 t(1;19)(q23;p13)

IGH-CEBP IGH-ID4 del(6)(q) Aberration of 9p

Standard risk

Aberration of 11q dup(1q) -7 dic(9;20)(p13;q11) dic(9;12)(p11-21;p11-13) Any other aberration normal karyotype t(9;22)(q34;q11) iAMP21

MLL translocations

High risk

„near“ haploidy (˂30 chromosomes) Low hypodiploidy (30-39 chromosomes) t(17;19)(q23;p13) Aberration of 17p Loss of 13q

Moorman et al., Lancet Oncol 2010

100%

t(12;21)

t(1;19) 50% t(4;11) t(9;22)

1

2

3

4

5

years

Today, karyotype remains the gold standard for classification of patients with childhood ALL into risk group for treatment.

ALL

Prognostic impact of specific chromosomal aberrations in childhood acute lymphoblastic leukemia (ALL)

I-FISH:

Conventional Cytogenetics:

B-ALL: „triple test“

BM direct/24h cultivation (childhood) 24h/48h cultivation (adults) at least 20 metaphases

ETV6/RUNX1 MLL Hyperdiploidy

or aberrant karyotype

T-ALL:

TCR genes targeted FISH or other molecular cytogenetic techniques (mFISH/mBAND, array CGH)

TCRαδ (14q11), TCRβ (7q34), TCRγ (7p14)

Report according to ISCN

TP16 (9p21) ABL1 (9q34)

ALL

Acute Lymphoblastic Leukemia (ALL)

CONCLUSIONS The impact of cytogenetic analyses on clinical diagnostics of hematological malignancies has increased dramatically during recent years → laboratory techniques have to be optimized to provide reliable results for optimal patient care. Quick and correct results save time and money by preventing unnecessary additional diagnostics and suboptimal treatment approaches. Standardization of cytogenetic diagnostic protocols may help to improve diagnosis, and hence treatment outcome of hematologic malignancies.